Project Summary/Abstract Efforts to characterize the HIV reservoir in patients have been limited by our inability to isolate viable cells containing HIV. Here we propose a novel method that uses CRISPR/Cas9 to genetically tag the latent HIV provirus. Provirus tagging will enable the isolation, study, and expansion of highly pure live HIV+ cells from research participants on a scale not previously possible. We will use this technique to investigate HIV integration sites, whole viral genomes, and T cell receptors of infected cells, thus providing new insights into the HIV reservoir and its maintenance. Proof of principle for this approach has been established in preliminary experiments, where we developed a tagging method that utilizes CRISPR/Cas9 and homology-independent targeted integration (HITI). This CAPTIV (CRISPR-assisted provirus tagging in vitro) method has been validated in ACH-2 cells, and allows insertion-dependent expression of a selectable marker from the provirus. Once isolated, CAPTIV-tagged cells grow normally in culture. Analysis of tagged cells using an HIV probe capture technique, which allows HIV sequence enrichment prior to NGS sequencing, enabled us to determine HIV integration sites and obtain full provirus sequences. In this grant we aim to use the CAPTIV technique in HIV+ participant samples so that we can perform a detailed genomic analyses of HIV+ CD4+ T cell clones. In Aim 1, we will first optimize the CAPTIV technique for use in primary CD4+ T cells infected with replication defective HIV by varying electroporation conditions, using AAV marker template donors, and modifying culture conditions. We aim to achieve a tag insertion efficiency of >10%. The optimal CAPTIV protocol will then be used to tag HIV+ CD4+ T cells obtained from research participants in the presence or absence of ART, so that HIV+ cells can be isolated for expansion and genetic analysis. In Aim 2, we will genetically analyze HIV+ CD4+ T cell clones or bulk pools of HIV+ CD4+ T cells expanded after CAPTIV isolation from participant samples. We will first use HIV probe capture and Illumina sequencing to identify HIV integration sites in bulk pools of HIV+ cells, and use this information to infer integration site preferences. We will then use HIV probe capture Illumina sequencing from individual HIV+ clones, or 10X Genomics sequencing data from bulk pools of HIV+ cells, to gain paired integration site and complete provirus sequence data. Finally, we will use HIV probe capture Illumina sequencing and gene- specific PCR to obtain integration sites, complete provirus sequences, and T cell receptor sequences from individual HIV+ clones. The overall goal of these studies is to perfect the isolation of gene-tagged HIV+ cells from participant samples, and to use this approach to gain new insights into the HIV reservoir and its maintenance during ART.